多胺生物合成的抑制对转化细胞c—Ha—ras癌基因表达的调控

抗多胺代谢剂－－二氟甲基鸟氨酸（ＤＦＭＯ）作用于经含点突变的Ｈａ－ｒａｓ基因片段转染的转化细胞（ＨＲ－１细胞）引起细胞生长的抑制,其抑制率随ＤＦＭＯ浓度的增加而增大,此时细胞多停滞于Ｇ１期;多胺合成的关键酶鸟氨酸脱羧酶（ＯＤＣ）活性显著下降;Ｈａ－ｒａｓ癌基因ｍＲＮＡ及ｒａｓＰ＾２１蛋白的表达受到抑制;而外源性腐胺与ＤＦＭＯ的同时加入可防止上述一系列改变的发生,说明ＤＦＭＯ使ＨＲ－１细胞某些表型向     

Forskolin对放射性转化细胞TGF基因及部分癌基因表达的影响

3~H-TdR放射性转化细胞(TC3H/10)经1×10~(-5)mol/L Foskolin处理24 h后,TGFα、c-myc、c-K-ras基因的mRNA表达下降,TGFβ、c-fos基因表达无明显变化。非转化细胞(NC3H/10)经相同条件处理,TGFα、TGFβ、c-myc及c-K-ras基因表达无显著改变。提示:Forskolin介导的转化细胞的生长抑制作用与TGFa、cmyc、c-K-ras基因的转录表达下降有关。     

多胺调控细胞生长机制的研究进展

多胺(腐胺、精脒、精胺)是真核细胞体内重要的多聚阳离子,对细胞的生长增殖发挥重要作用。快速生长的细胞内含有高浓度的多胺,降低多胺含量将抑制细胞的生长,阻滞细胞周期,而升高多胺含量则会促进细胞快速生长增殖。对多胺的调控已经成为研究细胞生长增殖调控的切入点之一。     

Forskolin对放射性转化细胞TGF基因及部分癌基因表达的影响

＾３Ｈ－ＴｄＲ放射性转化细胞经１×１０＾－５ｍｏｌ／Ｌ Ｆｏｓｋｏｌｉｎ处理２４ｈ后,ＴＧＦａ,ｃ－ｍｙｃ,ｃ－Ｋ－ｒａｓ基因的ｍＲＮＡ表达下降;ＴＧＦβ,ｃ－ｆｏｓ基因表达无明显变化。非转化细胞经相同条件处理,ＴＧＦａ,ＴＧＦβ,ｃ－ｍｙｃ及ｃ－Ｋ－ｒａｓ基因表达无显著改变。提示：Ｆｏｒｓｋｏｌｉｎ介导的转化细胞的生长抑制作用与ＴＧＦａ,ｃ－ｍｙｃ,ｃ－Ｋ－ｒａｓ基因的转录表达下降有关。     

r射线诱发大鼠胚胎细胞转化与N—ras的激活

以ｒ射线诱发转化的大鼠胚胎细胞（ＲＥＣ：ｃｙｃ：ｒ３３）的ＤＮＡ构建粘粒基因库,用总基因库ＤＮＡ转染ＮＩＨ／３Ｔ３细胞,产生转化灶的ＤＮＡ作二轮转染,二轮转化的ＮＩＨ３／Ｔ３细胞内有大鼠ＲＥＣ：ｍｙｃ：ｒ３３ＤＮＡ中具转化活性的Ｎ－ｒａｓ基因,用不对称ＰＣＲ和ＤＮＡ序列分析法证明,ＲＥＣ：ｍｙｃ：ｒ３３细胞中鼠Ｎ－ｒａｓ的活化是由于第６１位密码子的Ａ→Ｇ点空为。ＮＩＨ／３Ｔ３转化灶中鼠Ｎ－ｒａｓ也     

多胺参与细胞程序性死亡调控的研究进展

多胺被认为是影响细胞存活的一个关键分子。有证据显示,多胺可直接或间接参与细胞程序性死亡的调控。多胺与细胞程序性死亡直接相关,是指其参与特定的生物学过程及与导致细胞程序性死亡的分子/结构发生相互作用;间接相关,是指多胺通过调控细胞程序性死亡的代谢衍生物,如异化和互变产物来调控这一过程。此外,多胺代谢过程中的细胞毒性产物也参与到细胞程序性死亡的级联反应中。因此,对动植物中依赖于多胺的细胞程序性死亡的最新研究进展进行综述,可为进一步研究提供一些参考。     

Solubilization and stabilization of beta-carotene in niosomes: delivery to cultured cells

Carotenoids exhibit preventive effects against major diseases, including cancer and atherosclerosis. However, experimental studies on carotenoid functions in cultured cells are limited by the absence of an adequate method of solubilizing carotenoids, since they are unstable when exposed to light or oxygen and highly hydrophobic. In this study, we developed a niosomal formulation, consisting of non-ionic surfactants and cholesterol, which both solubilized and stabilized beta-carotene and that allowed to deliver it to cultured cells at concentrations spanning the range of physiological levels. beta-Carotene contained in niosomes was highly resistant to sunlight, high temperatures and oxidative stress induced by different sources of free radicals. The carotenoid was extremely stable in culture medium up to 96 h. Moreover, it was easily taken up by both immortalized and transformed cells at carotenoid concentrations which ranged from 0.1 to 2 microM. Therefore, niosomes provide a convenient, nontoxic and inexpensive vehicle for beta-carotene in cell culture.     

HL-60-AR白血病细胞诱导分化性状的持续性表达

HGPRT~-人早幼粒白血病细胞突变株(HL-60-AR)与RA保温一定时间后,洗去药物继续培养,细胞分化性状(NBT还原能力、细胞膜C_3补体受体及形态变化)不但继续存在,而且能持续表达。撤去RA后连续传代培养,至少在传三代后细胞分化性状仍高度表达。然而,DMSO对HL-60-AR细胞的作用特点明显不同于RA。HL-60-AR细胞分化伴随增殖能力的降低。核酸分子杂交结果表明,细胞c-myc癌基因表达受抑先于细胞分化性状的获得和增殖能力的下降。     

Catabolism of polyamines in the rat: Polyamines and their non-α-amino acid metabolites

The metabolic fate of stable isotopically labeled polyamines was investigated after their first and second intraperitoneal injection in rats. Using gas chromatographic and mass fragmentographic analyses of acid-hydrolyzed 24-h urines, some aspects of the polyamine metabolism could be elucidated. After the injections with hexadeutero-1,3-diaminopropane, obly labeled 1,3-diaminopropane was recovered from the urine samples. The rat injected with tetradeuteroputrescine excreted labeled putrescine excreted labeled putrescine, γ-amino-n-butyric acid, 2-hydroxyputrescine and spermidine, while the urine samples of the rat after the injections with tetradeuterocadaverine contained labeled cadaverine and δ-aminovaleric acid. The injections of hexadeuterospermidine led to the appearance of labeled spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, γ-amino-n-butyric acid, 1,3-diaminopropane, β-alanine and spermine. After the injections with octadeuterospermine, labeled spermine, N-(3-aminopropyl)-N′-(2-carboxyethyl)-1,4-diaminobutane, N,N′-bis(2-carboxyethyl)-1,4-diaminobutane, spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, 1,3-diaminopropane, β-alanine, 2-hydroxyputrescine and possibly γ-amino-n-butyric acid were recovered. Clear differences between the metabolism after the first and second injection were noted for putrescine, spermidine and spermine, which is suggestive for enzyme induction and/or the existence of salvage pathways.     

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.     

HDAC inhibitors induce apoptosis but not cellular senescence in Gadd45α-deficient E1A+Ras cells

HDAC inhibitors (HDIs) induce irreversible cell cycle arrest and senescence in E1A+Ras expressing cells. Furthermore, HDIs activate Gadd45α/NF-κB signaling pathway to suppress apoptosis thereby promoting the cell survival. Here, to clarify the role of Gadd45α in realization of the antiapoptotic program, we compared wild-type E1A+Ras cells and the cells with knockout of gadd45α gene (Gadd45α−/− cells). As in Gadd45α-expressing E1A+Ras cells, HDIs induce irreversible cell cycle arrest in Gadd45α−/− cells, but the arrested cells do not senesce and eventually die due to activation of the apoptotic death program. These data suggest that the expression of Gadd45α is involved in maintaining the balance of pro- and anti-apoptotic stimuli, while lack or loss of Gadd45 directs the cells to apoptosis after HDIs treatment. Appropriately Gadd45α-deficient cells demonstrate a higher level of pro-apoptotic signals, whereas the anti-apoptotic program is suppressed. The elevated apoptotic background of Gadd45α−/− cells is accompanied by higher levels of Ser15-phosphorylated p53 and p21/Waf1 proteins that additionally commit the cells to HDIs-induced apoptosis. Additionally, loss of Gadd45α protein activates the DDR signaling pathway as demonstrated by nuclear pATM staining, accumulation of γH2AX foci and an increase of single-strand DNA breaks. Thus, in wild-type E1A+Ras cells the p53-dependent expression of Gadd45α is necessary not only for DNA repair and HDI-induced cellular senescence, but also to withstand to apoptosis after DNA damage and stress. Therefore the use of HDIs in combination with agents that block Gadd45α function may have promise for cancer therapy.     

Synthesis and evaluation of N8-acetylspermidine analogues as inhibitors of bacterial acetylpolyamine amidohydrolase

Polyamines are small essential polycations involved in many biological processes. Enzymes of polyamine metabolism have been extensively studied and are attractive drug targets. Nevertheless, the reversible acetylation of polyamines remains poorly understood. Although eukaryotic N⁸-acetylspermidine deacetylase activity has already been detected and studied, the specific enzyme responsible for this activity has not yet been identified. However, a zinc deacetylase from Mycoplana ramosa, acetylpolyamine amidohydrolase (APAH), has been reported to use various acetylpolyamines as substrates. The recently solved crystal structure of this polyamine deacetylase revealed the formation of an ‘L’-shaped active site tunnel at the dimer interface, with ideal dimensions and electrostatic properties for accommodating narrow, flexible, cationic polyamine substrates. Here, we report the design, synthesis, and evaluation of N⁸-acetylspermidine analogues bearing different zinc binding groups as potential inhibitors of APAH. Most of the synthesized compounds exhibit modest potency, with IC₅₀ values in the mid-micromolar range, but compounds bearing hydroxamate or trifluoromethylketone zinc binding groups exhibit enhanced inhibitory potency in the mid-nanomolar range. These inhibitors will enable future explorations of acetylpolyamine function in both prokaryotes and eukaryotes.     

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium.

Abbreviations: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion     

无血清生长的化学转化细胞产生并应答转化生长因子

BA10-IR细胞系是7-甲基苯蒽[7-MethylBenz(a)Anthracene(7-MBA)]转化的叙利亚地鼠胚成纤维细胞。该细胞克隆化后分离出一株在无血清无外源分裂因子培养基中生长的亚系——BA10—IR S~-/M~-。BA10-IR S~-/M~-细胞在无血清无外源分裂因子培养基中产生转化生长因子(TGFs),并刺激指示细胞在半固体培养基中着壁不依赖生长。同样BA10—IR S~-/M~-细胞所释放的TGFs能加强BA10-IR S~-/M~-本身无血清着壁不依赖生长能力。这可能正是BA10-IR S~-/M~-细胞能在无血清条件下长期生长的原因。自身分泌TGFs,又刺激自身生长,形成了一个自发生长调节系统。